感謝大家回答 但我還是有點疑問 摘錄自上篇引文 http://www.bio.net/bionet/mm/microbio/1997-May/009308.html 1) check the pellet to see if you are really solubilizing the protein. Try conditions like 50mm, 250mm, 1M NaCl, with/without detergent, with/without betamercaptoethanol (do NOT use DTT - it binds strongly to the NI column) →這個意思是說用高鹽或清潔劑、還原劑等去把pellet上面的蛋白溶到sup嗎？ 2)do not use DTT or EDTA if can avoid it. If you must use it to resolubilize, then wash by diluting out, and spin several times before loading onto the column. 3)try phosphate buffer or lower strength TRIS or HEPES for loading. In some cases amines, including the buffers, can interact with the column and prevent binding. 4)If you can't get the protein off the column, supplement with imadazole, and try additional NaCl or detergent or glycerol to get the bound protein to elute. 5)Try batch elution: empty the colum into a tube that can be gently rocked, filter off the resin in the morning, measure protein in the solution. 6)Presence of DNA or macromolecules may inhibit proper binding. Some people treat with a small amount of DNase to chop up DNA into small pieces. High viscosity is a problem. Large particulates, aggregates of proteins should be spun out before loading. 7)If your protein is simply not soluble, then try putting your tag on the opposite end, i.e. switch N term for C-term tag. Sometimes the end which the tag is on makes a big difference in solubility. 8)As a last resort, if the protein simply will not elute from the resin, strip the column with EDTA. This strips the NI plus the protein, but you should be able to measure the eluate for protein content---it just has a lot of NI in it. And the column can be regenerated. →可是上面不是說盡量避免使用EDTA？ 這樣會不會造成蛋白變性？因為我要用來做EMSA，所以盡量想用native elution 還有連同Ni一起strip下來會不會很難去除Ni？ 9)The protein structure can be 'loosened - up' in 1 to 2 M urea or guanidine, which really does not denature. Maybe even load the column in this. But when proteins have been totally denatured then everything tends to bind to the NI column. →這個方式是說讓蛋白"稍微變性"囉？ 因為我懷疑我的蛋白已經不只用His去抓Ni了 它是整個緊緊抓住才會elute不太下來 所以想試試這個方法 但是又怕不小心變性了的蛋白失去活性 --

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